Gene replacement for Duchenne muscular dystrophy (DMD) with micro-dystrophins has entered clinical trials, but efficacy on preventing heart failure is unknown. Although most DMD patients die from heart failure, cardiomyopathy is undetectable until the teens so efficacy from trials in young boys will be unknown for a decade. Available DMD animal models were sufficient to demonstrate micro-dystrophin efficacy on earlier onset skeletal muscle pathology underlying loss of ambulation and respiratory insufficiency in patients. However, no mouse models progressed into heart failure and dog models show highly variable progression insufficient to evaluate efficacy of micro-dystrophin or other therapies on DMD heart failure. To overcome this barrier, we have generated the first DMD mouse model that reproducibly progresses into heart failure. This model shows cardiac inflammation and fibrosis occur prior to reduced function. Fibrosis does not continue to accumulate, but inflammation persists after function declines. We used this model to test micro-dystrophin gene therapy efficacy on heart failure prevention for the first time. Micro-dystrophin prevents declines in cardiac function and prohibits onset of inflammation and fibrosis. This model will allow identification of committed pathogenic steps to heart failure and testing of genetic and non-genetic therapies to optimize cardiac care for DMD patients.
Zachary M. Howard, Lisa E. Dorn, Jeovanna Lowe, Megan D. Gertzen, Pierce C. Ciccone, Neha Rastogi, Guy L. Odom, Federica Accornero, Jeffrey S. Chamberlain, Jill A. Rafael-Fortney
Altered inflammation and tissue remodeling are cardinal features of cardiovascular disease and cardiac transplant rejection. Neutrophils have increasingly been understood to play a critical role in acute rejection and early allograft failure; however, discrete mechanisms that drive this damage remain poorly understood. Herein, we demonstrate that early acute cardiac rejection increases allograft prolyl endopeptidase (PE) in association with de novo production of the neutrophil pro-inflammatory matrikine proline-glycine-proline (PGP). In a heterotopic murine heart transplant model, PGP production and PE activity were associated with early neutrophil allograft invasion and allograft failure. Pharmacologic inhibition of PE with Z-Pro-Prolinal reduced PGP, attenuated early neutrophil graft invasion, and reduced pro-inflammatory cytokine expression. Importantly, these changes helped preserve allograft rejection-free survival and function. Notably, within two independent patient cohorts, both PGP and PE activity were increased among patients with biopsy-proven rejection. The observed induction of PE and matrikine generation provides a novel link between neutrophilic inflammation and cardiovascular injury, represents a potentially new target to reduce allogenic immune responses, and uncovers a previously unrecognized mechanism of cardiovascular disease.
Gregory A. Payne, Nirmal S. Sharma, Charitharth V. Lal, Chunyan Song, Lingling Guo, Camilla Margaroli, Liliana Viera, Siva Kumar, Jindong Li, Melanie Bosley, Dongqi Xing, Xin Xu, J. Michael Wells, James F. George, Jose A. Tallaj, Massoud Leesar, J. Edwin Blalock, Amit Gaggar
A hallmark of impaired myocardial energetics in failing hearts is the downregulation of the creatine kinase (CK) system. In heart failure patients and animal models, myocardial phosphocreatine content and the flux of the CK reaction are negatively correlated with the outcome of heart failure. While decreased CK activity is highly reproducible in failing hearts, the underlying mechanisms remains elusive. Here, we report an inverse relationship between the activity and acetylation of CK muscle form (CKM) in human and mouse failing hearts. Hyperacetylation of recombinant CKM disrupted MM homodimer formation and reduced enzymatic activity, which could be reversed by sirtuin 2 treatment. Mass spectrometry analysis identified multiple lysine residues on the MM dimer interface, which were hyperacetylated in the failing hearts. Molecular modeling of CK MM homodimer suggested that hyperacetylation prevented dimer formation through interfering salt bridges within and between the 2 monomers. Deacetylation by sirtuin 2 reduced acetylation of the critical lysine residues, improved dimer formation, and restored CKM activity from failing heart tissue. These findings reveal a potentially novel mechanism in the regulation of CK activity and provide a potential target for improving high-energy phosphoryl transfer in heart failure.
Matthew A. Walker, Juan Chavez, Outi Villet, Xiaoting Tang, Andrew Keller, James E. Bruce, Rong Tian
Preterm birth increases the risk for pulmonary hypertension and heart failure in adulthood. Oxygen therapy can damage the immature cardiopulmonary system and may be partially responsible for the cardiovascular disease in adults born preterm. We previously showed that exposing newborn mice to hyperoxia causes pulmonary hypertension by 1 year of age that is preceded by a poorly understood loss of pulmonary vein cardiomyocyte proliferation. We now show that hyperoxia also reduces cardiomyocyte proliferation and survival in the left atrium and causes diastolic heart failure by disrupting its filling of the left ventricle. Transcriptomic profiling showed that neonatal hyperoxia permanently suppressed fatty acid synthase (Fasn), stearoyl-CoA desaturase 1 (Scd1) and other fatty acid synthesis genes in the atria of mice, the HL-1 line of mouse atrial cardiomyocytes and left atrial tissue explanted from human infants. Suppressing Fasn or Scd1 reduced HL-1 cell proliferation and increased cell death while overexpressing these genes maintained their expansion in hyperoxia, suggesting oxygen directly inhibits atrial cardiomyocyte proliferation and survival by repressing Fasn and Scd1. Pharmacologic interventions that restore Fasn, Scd1 and other fatty acid synthesis genes in atrial cardiomyocytes may thus provide a way of ameliorating the adverse effects of supplemental oxygen on preterm infants.
Ethan David Cohen, Min Yee, George A. Porter, Jr., Erin E. Ritzer, Andrew N. McDavid, Paul S. Brookes, Gloria S. Pryhuber, Michael A. O'Reilly
Myotonic dystrophy type 1 (DM1) is caused by a CTG-repeat expansion in the DMPK gene. Expression of pathogenic expanded CUG-repeat (CUGexp) RNA causes multisystemic disease by perturbing the functions of RNA binding proteins, resulting in expression of fetal protein isoforms in adult tissues. Cardiac involvement affects 50% of individuals with DM1 and causes 25% of disease-related deaths. We developed a transgenic mouse model for tetracycline-inducible and heart-specific expression of human DMPK mRNA containing 960 CUG repeats. CUGexp RNA is expressed in atria and ventricles and induced mice exhibit electrophysiological and molecular features of DM1 disease including cardiac conduction delays, supraventricular arrhythmias, nuclear RNA foci with Muscleblind protein colocalization and alternative splicing defects. Importantly, these phenotypes were rescued upon loss of CUGexp RNA expression. Transcriptome analysis revealed gene expression and alternative splicing changes in ion transport genes that are associated with inherited cardiac conduction diseases, including a subset of genes involved in calcium handling. Consistent with RNA-seq results, calcium handling defects were identified in atrial cardiomyocytes isolated from mice expressing CUGexp RNA. These results identify potential tissue-specific mechanisms contributing to cardiac pathogenesis in DM1 and demonstrate the utility of reversible phenotypes in our model to facilitate development of targeted therapeutic approaches.
Ashish N. Rao, Hannah M. Campbell, Xiangnan Guan, Tarah A. Word, Xander H.T. Wehrens, Zheng Xia, Thomas A. Cooper
Background: Loss-of-function variants in SCN1B, encoding voltage-gated sodium channel β1 subunits, are linked to human diseases with high risk of sudden death, including epileptic encephalopathy and cardiac arrhythmia. β1 subunits modulate the cell-surface localization, gating, and kinetics of sodium channel pore-forming a subunits. They also participate in cell-cell and cell-matrix adhesion, resulting in intracellular signal transduction, promotion of cell migration, calcium handling, and regulation of cell morphology. Methods: We investigated regulated intramembrane proteolysis (RIP) of β1 by BACE1 and γ-secretase.Results: We show that β1 subunits are substrates for sequential RIP by BACE1 and γ-secretase, resulting in the generation of a soluble intracellular domain (ICD) that is translocated to the nucleus. Using RNA-seq, we identified a subset of genes that are downregulated by β1-ICD overexpression in heterologous cells but upregulated in Scn1b null cardiac tissue which, by definition, lacks β1-ICD signaling, suggesting that the β1-ICD may normally function as a molecular brake on gene transcription in vivo. Conclusion: We propose that human disease variants resulting in SCN1B loss-of-function cause transcriptional dysregulation that contributes to altered excitability. These results provide important new insights into the mechanism of SCN1B-linked channelopathies, adding RIP-excitation coupling to the multi-functionality of sodium channel β1 subunits.
Alexandra A. Bouza, Nnamdi Edokobi, Samantha L. Hodges, Alexa M. Pinsky, James Offord, Lin Piao, Yan-Ting Zhao, Anatoli N. Lopatin, Luis F. Lopez-Santiago, Lori L. Isom
The pathogenesis of preeclampsia and other hypertensive disorders of pregnancy remains poorly-defined despite the substantial burden of maternal and neonatal morbidity associated with these conditions. In particular, the role of genetic variants as determinants of disease susceptibility remains unknown. Storkhead-box protein 1 (STOX1) was first identified as a preeclampsia risk gene through family-based genetic linkage studies in which loss-of-function variants were proposed to underlie increased preeclampsia susceptibility. We generated a genetic Stox1 loss-of-function mouse model (Stox1 KO), to evaluate whether STOX1 regulates blood pressure in pregnancy. Pregnant Stox1 KO mice developed gestational hypertension evidenced by a significant increase in blood pressure compared with wild type by E17.5. While severe renal, placental, or fetal growth abnormalities were not observed, the Stox1 KO phenotype was associated with placental vascular and extracellular matrix abnormalities. Mechanistically, we found that gestational hypertension in Stox1 KO mice resulted from activation of the uteroplacental renin-angiotensin system. We confirmed this mechanism by showing that treatment of pregnant Stox1 KO mice with an angiotensin II receptor blocker rescued the phenotype. Our study demonstrates the utility of genetic mouse models for uncovering links between genetic variants and effector pathways implicated in the pathogenesis of hypertensive disorders of pregnancy.
Jacqueline G. Parchem, Keizo Kanasaki, Soo Bong Lee, Megumi Kanasaki, Joyce L. Yang, Yong Xu, Kadeshia M. Earl, Rachel A. Keuls, Vincent H. Gattone, Raghu Kalluri
Cardiopulmonary bypass (CPB) is required during most cardiac surgeries. CBP drives systemic inflammation and multi-organ dysfunction that is more severe in neonatal patients. Limited understanding of molecular mechanisms underlying CPB-associated inflammation presents a significant barrier to improving clinical outcomes. To better understand these clinical issues, we performed the first mRNA-sequencing on total circulating leukocytes from neonatal patients undergoing CPB. These data identified myeloid cells, particularly monocytes, as the major cell type driving transcriptional responses to CPB. Furthermore, Interleukin-8 (IL8) and Tumor Necrosis Factor-α (TNFα) were inflammatory cytokines robustly upregulated in leukocytes from both patients and piglets exposed to CPB. To delineate a molecular mechanism, we exposed THP-1 human monocytic cells to CPB-like conditions including artificial surfaces, high shear stress, and cooling/rewarming. Here, shear stress was found to drive cytokine upregulation via calcium-dependent signaling pathways. We also observed a subpopulation of THP-1 cells died via TNFα-mediated necroptosis in our model, which we hypothesize contributes to post-CPB inflammation. Together, our study identifies a shear-stress modulated molecular mechanism that drives systemic inflammation in pediatric CPB patients. These are also the first data to demonstrate that shear-stress causes necroptosis. Finally, we observe that calcium and TNFα signaling are novel targets to ameliorate post-CPB inflammation.
Lan N. Tu, Lance Hsieh, Masaki Kajimoto, Kevin Charette, Nataliya Kibiryeva, Adriana Forero, Sarah E. Hampson, Jennifer Marshall, James O’Brien, Marta Scatena, Michael A. Portman, Ram Savan, Christopher Benner, Alberto Aliseda, Muhammad Nuri, Douglas Bittel, Peter Pastuszko, Vishal Nigam
Cardiac fibrosis is a pathophysiologic hallmark of the aging heart. In the uninjured heart, cardiac fibroblasts exist in the quiescent state, but little is known about how proliferation rates and fibroblast transcriptional programs change throughout the lifespan of the organism from the immediate postnatal period to adult life and old age. Using EdU pulse labeling, we demonstrate that more than 50% of cardiac fibroblasts are actively proliferating in the first day of post-natal life. However, within 4 weeks of birth in the juvenile animal, only 10% of cardiac fibroblasts are proliferating. By early adulthood, the fraction of proliferating cardiac fibroblasts further decreases to approximately 2%, where it so remains throughout the rest of the organism’s life span. Examination of absolute cardiac fibroblast numbers demonstrated concordance with age related changes in fibroblast proliferation with no significant differences in absolute cardiac fibroblast numbers between animals 14 weeks and 1.5 years of age. We demonstrate that the maximal changes in cardiac fibroblast transcriptional programs and in particular collagen expression occur within the first weeks of life from the immediate postnatal to the juvenile period. We show that even though the aging heart exhibits an increase in the total amount of accumulated collagen, transcription of various collagens and ECM genes both in the heart and cardiac fibroblast is maximal in the newly born and juvenile animal and decreases with organismal aging. Examination of DNA methylation changes both in the heart and in cardiac fibroblasts did not demonstrate significant changes in differentially methylated regions between young and old mice. Our observations demonstrate that cardiac fibroblasts attain a stable proliferation rate and transcriptional program early in the life span of the organism and suggest a model of cardiac aging where phenotypic changes in the aging heart are not directly attributable to changes in proliferation rate or altered collagen expression in cardiac fibroblasts.
Rimao Wu, Feiyang Ma, Anela Tosevska, Colin Farrell, Matteo Pellegrini, Arjun Deb
Atrial fibrillation (AF) commonly occurs after surgery and is associated with atrial remodeling. TRPV4 is functionally expressed in the heart, and its activation affects cardiac structure and functions. We hypothesized that TRPV4 blockade alleviates atrial remodeling and reduces AF induction in sterile pericarditis (SP) rats. TRPV4 antagonist GSK2193874 or vehicle was orally administered 1 d before pericardiotomy. AF susceptibility and atrial function were assessed using in vivo electrophysiology, ex vivo optical mapping, patch-clamp, and molecular biology on day 3 after surgery. TRPV4 expression increased in the atria of SP rats and patients with AF. GSK2193874 significantly reduced AF vulnerability in vivo and the frequency of atrial ectopy and AF with a reentrant pattern ex vivo. Mechanistically, GSK2193874 reversed the abnormal action potential duration (APD) prolongation in atrial myocytes through the regulation of voltage-gated K+ currents (IK), reduced the activation of atrial fibroblasts by inhibiting P38, AKT, and STAT3 pathways, and alleviated the infiltration of immune cells. Our results reveal that TRPV4 blockade prevents abnormal changes in atrial myocyte electrophysiology and ameliorated atrial fibrosis and inflammation in SP rats, and, therefore, might be a promising strategy to treat AF, particularly post-operative AF.
Jie Liao, Qiongfeng Wu, Cheng Qian, Ning Zhao, Zhaoyang Zhao, Kai Lu, Shaoshao Zhang, Qian Dong, Lei Chen, Qince Li, Yimei Du
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